We have been studying crystallins, the abundant water-soluble proteins responsible for the optical properties of the eye lens, as a model for tissue-specific gene expression. Crystallins are especially interesting since they are also often expressed in non-lens tissues at lower concentrations where they have enzymatic or other non-refractive functions. Within this fiscal year we have completed to publication studies initiated earlier. These have shown that that the proximal TATA box of the mouse alphaB-crystallin gene is used preferentially for lens expression but is not responsible for lens-specificity while the upstream TATA sequence does have a role in lens-specific expression, that the two lens-specific cis-control elements (LSR1 and 2) of the alphaB-crystallin gene are activated by Pax- 6 and retinoic acid receptors, and that Pax-6 binds to and represses an activating element (PL-2) of the chicken betaB1- crystallin gene. We have also shown that retinoblastoma protein (pRB) and the TATA box-binding protein (TBP) interact with Pax-6 via its homeodomain, suggesting overlapping pathways for Pax-6 during lens fiber cell differentiation. Several years ago we showed that both the alphaA and alphaB-crystallins had the ability to undergo limited serine-specifc, magnesium-dependent, cyclic AMP-independent autophosphorylation in addition to its known cyclic AMP-dependent phosphorylation. We now have shown that serine-specific, cyclic AMP-independent autophosphorylation extends to betaB2-crystallin; this polypeptide shares with alpha- crystallins the properties of being also phosphorylated by a cyclic AMP-dependent process and being expressed outside of the lens. We have reviewed these studies and have suggested that these crystallins may be involved in yet to be discovered signal transduction pathways. This would open a new area of investigation for these ubiquitious crystallins. We have also examined the pattern of expressed genes in epithelia from normal and cataractous human lenses has been examined by differential display. The results revealed 3 mRNAs with higher expression and 12 with lower expression in the cataractous lenses. Protein phosphatase 2A regulatory subunit mRNA, a mitotic suppressor, was lowered, while metallotheonein Iia, a detoxification protein, was increased. Studies on the heredity mouse Cat2nop (mutation in gammaB-crystallin) and Cat2t (mutation in gammaE- crystallin) cataracts have indicated that the protein Bax is upregulated, linking crystallins with cell cycle regulation. Studies on jellyfish and scallops, both species with remarkably sophisticated eyes with cellular lenses, have continued with the goal of understanding the evolutionary origins of vertebrate and ultimately human eyes. This year we have completed and published a study demonstrating that jellyfish have an RXR gene whose product is expressed during development, binds 9-cis retinoic acid and can bind to the jellyfish J1-crystallin promoters. This is the first demonstration of an RXR in diploblastic species and suggests that regulation of crystallin gene expression by retinoid signalling may extend to the invertebrates. Finally, we have shown that the scallop lens has one major crystallin that is closely related to aldehyde dehydrogenase1/2. It lacks enzymatic activity with all substrates tested to date and when purified exists as a dimer. The presence of this enzyme-related cyrstallin in scallops extends the existence of enzyme- crystallins to these primitive invertebrates.